The isolation, characterization and manipulation of nucleic acids has numerous present or potential applications, including those in the basic research, diagnostic and forensic fields. Valuable information about gene expression in in vivo, in situ, and in vitro systems can be obtained by monitoring the abundance of the mRNA encoded by those genes. Methods involving the synthesis of cDNA from mRNA have also enhanced the study of gene expression, for example, by facilitating gene cloning and the production of desired recombinant proteins.
With existing methods for the study or use of mRNA and cDNA, one problematic scenario can arise where the sample size is small, or the relative abundance of an individual mRNA or cDNA species in a sample is low. In such situations, where the availability or accessibility of the desired mRNA or cDNA is compromised (or their amounts are otherwise limited), the lower limits of monitoring or manipulation systems may be exceeded, thus leaving the desired mRNA or cDNA undetected, unrecoverable or unworkable. Therefore, the amplification of such mRNA and cDNA is an important molecular biology methodology, with particular significance in facilitating the detection and study of a broader range of mRNA molecules, and the isolation and manipulation of mRNA available in only minute quantities.
Known nucleic acid amplification methods may typically involve multiple steps and varying reaction conditions such as organic extraction and precipitation. As result, these methodologies can be labor intensive and costly. For example, to amplify RNA in a heterogenous RNA population, known methods can require that a second strand cDNA synthesis is performed in a volume that is more than three times the volume of the first strand cDNA synthesis reaction. Prior to the in vitro transcription (IVT) reaction, in which RNA polymerase may use promoter containing double-stranded cDNA as templates for RNA transcription, the cDNA sample typically must be cleaned by, for example, phenol extraction and ethanol precipitation. When these cleaning and concentration steps are not used, the IVT reaction may be inhibited due to undesirable buffer conditions and enzymatic activities.
In addition, although methods exist for the amplification of nucleic acids, they generally suffer from a phenomenon known as biased amplification. In these cases, the amplified population does not proportionally represent the population of nucleic acid species existing in the original sample. This drawback may preclude meaningful or reliable conclusions regarding the absolute amount or relative abundance of a desired nucleic acid species in the tested sample.
One common problem encountered by past amplification methods is the preference for the amplification of shorter nucleic acid templates. The enzymes responsible for the production of complements or copies of the nucleic acid templates (e.g., DNA and RNA polymerases, or reverse transcriptases) achieve such synthesis through a sequential, oriented process, whether 5′ to 3′ or 3′ to 5′. The probability that such an enzyme will complete a copying event thus may be greater with nucleic acid templates of shorter length. Accordingly, in a sample population containing nucleic acid templates of variable lengths, longer templates may be less likely than shorter templates to be amplified in complete, full-length form. This can result in a bias in the amplified population in favor of nucleotide sequences proximal to the 3′ poly(A) tail of mRNA, for example, a phenomenon known as 3′-sequence bias.
The synthesis of longer templates can also be difficult or less efficient due to interference from secondary and tertiary structure in the template. For example, with respect to nucleic acid amplification based on polymerase chain reaction (PCR) methodologies, longer templates in a sample may be under-represented in the amplified product if respective primers cannot anneal to begin another round of copying because the first round did not proceed to completion. Other potential sources of bias can reflect relative differences between longer and shorter templates. For example, longer templates may (i) not denature sufficiently, or (ii) have a greater likelihood of mismatches, and thus error propagation through amplification, but (iii) have an ability to anneal more easily.
The foregoing shows a need for methods and products involving the amplification of nucleic acids in a simplified manner, and preferably, to facilitate the preservation of the relative abundance of the individual nucleic acid species existing in the original sample.